Normal hemostasis is the result of a complex balance between the processes of clot formation (blood coagulation) and clot dissolution (fibrinolysis). The complex interactions between blood cells, specific plasma proteins and the vascular surface maintain the fluidity of blood unless injury and blood loss occur. Many significant disease states are related to abnormal hemostasis. Abnormal thrombus formation occurring in the coronary arterial vasculature due to the rupture of an established atherosclerotic plague is the major cause of acute myocardial infarction and unstable angina. Treatment of occlusive coronary thrombus by either thrombolytic therapy or percutaneous transluminal angioplasty is often accompanied by an acute thrombotic reclosure of the affected vessel which requires immediate resolution. A high percentage of patients undergoing major surgery in the lower extremities or the abdominal area suffer from thrombus formation in the venous vasculature which can result in reduced blood flow to the affected extremity and a predisposition to pulmonary embolism. Disseminated intravascular coagulopathy commonly occurs during septic shock, certain viral infections and cancer and is characterized by the rapid consumption of coagulation factors and systemic coagulation which results in the formation of life-threatening thrombi occurring throughout the vasculature leading to wide-spread organ failure.
Blood coagulation is the culmination of a series of amplified reactions in which several specific zymogens of serine proteases in plasma are activated by limited proteolysis. This results in the formation of an insoluble fibrin matrix which is required for the stabilization of the primary hemostatic plug. The interaction and propagation of the activation reactions occurs through the extrinsic and intrinsic pathways of coagulation as reviewed by Mackie, I. J. and Bull, H. A., "Normal Hemostasis and its Regulation" Blood Reviews, 3: 237-250 (1989). Both pathways are highly inter-dependent and converge in the formation of the serine protease factor xa from its zymogen, factor X. Factor Xa catalyzes the penultimate step in the blood coagulation cascade which is the formation of the serine protease thrombin. Thrombin goes on to cleave soluble fibrinogen in the plasma to form insoluble fibrin.
The biochemical and physiological characterization of factor X has been reviewed by Steinberg, M. and Nemerson, Y., "Activation of Factor X", Hemostasis and Thrombosis, First Edit., pp 91-111 (Colman, R. et. al. eds. 1982) and Mann, K. G. et. al., "Surface-Dependent Reactions of the Vitamin K-Dependent Enzyme Complexes", Blood, 76: 1-16 (1990). Human factor X circulates in plasma at a concentration of 170 nM. The enzyme is a two-chain glycoprotein containing 442 amino acid residues having an overall molecular size (Mr) of 59,000 as determined by sedimentation equilibrium centrifugation and approximately 67,000 by sodium dodecyl sulfate electrophoresis. DiScipio et. al., "A comparison of human prothrombin, Factor IX (Christmas Factor), Factor X (Stuart Factor) and Protein S", Biochemistry, 16: 698-706 (1977) and Leyfus et. al., "Characterization of a cDNA coding for human Factor X", Proc. Natl. Acad. Sci. USA, 82: 3699 (1984). Human factor X contains a light-chain subunit containing 139 amino acid residues (Mr=16,200) and a heavy chain subunit containing 303 amino acid residues (Mr=42,000) linked together by a single disulfide bond. The light chain of human factor X contains 11 glutamic acid residues which have been post-translationally modified to .gamma.-carboxy-glutamic acid and one asparagine acid moiety modified to .beta.-hydroxy aspartic acid. The heavy chain of factor X contains all of the glycosylated residues (15% overall) as well as the catalytic domain of the molecule.
Proteolytic activation of zymogen factor X to its catalytically active form, factor Xa, can occur by either the intrinsic or extrinsic coagulation pathways. The intrinsic pathway is referred to as intrinsic because everything needed for clotting is in the blood. Saito, H., "Normal Hemostatic Mechanisms", Disorders of Hemostasis, pp. 27-29, Grune & Stratton, Inc. (O. D. Ratnoff, M.D. and C. D. Forbes, M.D. edit. 1984). This pathway is comprised of the zymogen serine proteases, factors IX and XI, and the non-enzymatic co-factor, factor VIII. The initiation of the intrinsic pathway results in the activation of factor XI to XIa. Factor XIa catalyzes the activation of factor IX to factor IXa which in combination with the activated form of factor VIII on an appropriate phospholipid surface, results in the formation of the tenase complex. This complex also catalyzes the formation of the serine protease, factor Xa, from its zymogen, factor X, which subsequently results in clot formation.
The extrinsic pathway is referred to as extrinsic because the tissue factor which binds to and begins activation of factor VII comes from outside the blood. Saito, Id, The major components of this pathway are the zymogen serine protease, factor VII, and the membrane bound protein, tissue factor. The latter serves as the requisite non-enzymatic co-factor for this enzyme. The initiation of this pathway is thought to be an autocatalytic event resulting from the activation of zymogen factor VII by trace levels of activated factor VII (factor VIIa), both of which are bound to newly exposed tissue factor on membrane surfaces at sites of vascular damage. The factor VIIa/tissue factor complex directly catalyzes the formation of the serine protease, factor Xa, from its zymogen, factor X. Exposure of blood to injured tissue initiates blood clotting by the extrinsic pathway.
Proteolytic activation of zymogen factor X to its catalytically active form, factor Xa, results in the liberation of a 52 amino acid activation peptide from the amino-terminus of the heavy chain subunit. The intrinsic activation reaction is catalyzed by factor Ixa in a macromolecular complex with the non-enzymatic co-factor, factor VIII. Factor Xa formation via the extrinsic pathway is catalyzed by the catalytic complex of factor VIIa and tissue factor. Both of these reactions must occur on an appropriate phospholipid surface in the presence of calcium ions. The active product formed following either intrinsic or extrinsic activation of factor X is .alpha.-factor Xa. A second proteolytic cleavage which is thought to be autocatalytic, results in the formation of .beta.-factor Xa following the release of a 14 amino acid peptide from the carboxy-terminus of the heavy chain. Both forms of the activated molecule have the same catalytic activity as measured by their ability to promote coagulation in plasma or hydrolyze a peptidyl chromogenic substrate.
The formation of thrombin is catalyzed by factor Xa following the assembly of the catalytic prothrombinase complex as reviewed by Mann, K. G. et. al., "Surface-Dependent Reactions of the Vitamin K-Dependent Enzyme Complexes", Blood, 76: 1-16 (1990). This complex is composed of factor xa, the non-enzymatic co-factor Va and the substrate prothrombin all assembled on an appropriate phospholipid surface. The requirement of a macromolecular complex for efficient catalysis results in the protection of factor Xa from natural anticoagulant mechanisms such as heparin-antithrombin III mediated inhibition. Teite, J. M. and Rosenberg, R. D., "Protection of Factor Xa from neutralization by the heparin-antithrombin complex", J. Clin. Invest., 71: 1383-1391(1983). In addition sequestration of factor Xa in the prothrombinase complex also renders it resistant to inhibition by exogenous heparin therapy which also requires antithrombin III to elicit its anticoagulant effect.
Several examples of naturally occurring polypeptide inhibitors of factor Xa have been reported to have excellent specificity and potency. U.S. Pat. No. 4,588,587 to Gasic describes the anticoagulant activity of Haementeria offcinalis leech saliva. A principal component of the leech saliva, Antistasin was said to inhibit factor xa. See, Tuszynski, G. P. et. al., "Isolation and characterization of antistasin, an inhibitor of metastasis and coagulation", J. Biol. Chem., 262: 9718-9723 (1987); Nutt, E. et. al.,"The amino acid sequence of antistasin, a potent inhibitor of Factor xa reveals a repeated internal structure", J. Biol. Chem., 63: 10162-10167 (1988); Dunwiddie, C. et. al., "Antistasin, a leech-derived inhibitor of factor Xa, kinetic analysis of enzyme inhibition and identification of the reactive site", J. Biol. Chem., 264:16694-16699 (1989); and Han, J. H. et. al., "Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties", Gene, 75: 47-57 (1989).
A polypeptide reported to be a selective and potent inhibitor of factor Xa was originally isolated from whole body extracts of the soft tick Ornithidoros moubata. See Waxman, L. et. al., "Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa", Science, 248: 593-596(1990); Neeper M. P. et al.,"Characterization of recombinant tick anticoagulant peptide, a highly selective inhibitor of blood coagulation, factor Xa", J. Biol. Chem., 265: 17746-17752 (1990); and Jordan, S. P. et. al., "Tick anticoagulant peptide: kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa", Biochemistry, 29: 11095-11100 (1990); and Vlasuk et al., U.S. Pat. No. 5,239,058 (Aug. 24, 1993).
Plasma has been reported to contain a common inhibitor of both factor Xa and factor VIIa-tissue factor complex called lipoprotein-associated coagulation inhibitor (LACI). LACI is reported to consist of 276 amino acids and has been reported to inhibit the proteolytic activity of factor Xa directly, and in a factor Xa-dependent manner, factor VIIa-tissue factor complex. Girard, T. J. et al., Nature, 338: 518-520 (1989).
Other polypeptide inhibitors of factor Xa have also been reported. See, e.g., Jacobs, J. W. et. al., "Isolation and characterization of a coagulation factor Xa inhibitor from Black fly salivary glands", Thromb. Haemostas., 64: 235-238 (1990); Condra, C. et. al., "Isolation and structural characterization of a potent inhibitor of coagulation factor Xa from the leech Haementeria ghilianii", Thromb. Haemost., 61: 437-441 (1989); Brankamp, R. G. et. al., "Ghilantens: anticoagulants, antimetastatic proteins from the South American leech Haementeria ghilianii", J. Lab. Clin. Med., 115: 89-97 (1990); Blankenship, D. T. et. al., "Amino acid sequence of ghilanten: anti-coagulant-antimetastatic principle of the South American leech, Haementeria ghilianii"", Biochem. Biophys. Res. Commun., 166: 1384-1389 (1990); and Rigbi, M. et. al.,"Bovine factor Xa inhibiting factor and pharmaceutical compositions containing the same", European Patent Application, publication no. 352,903 (1990).
In addition to the above polypeptide inhibitors of factor Xa, small molecule inhibitors of this enzyme have been reported. See, Kam, et. al., "Mechanism based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants", Biochemistry, 27: 2547-2557 (1988); Tidwell, R. R. et. al., "Strategies for anticoagulation with synthetic protease inhibitors. Xa inhibitors versus thrombin inhibitors", Thromb. Res., 19: 339-349 (1980); Hitomi, Y. et. al., "Inhibitory effect of a new synthetic protease inhibitor (FUT-175) on the coagulation system", Haemostasis, 15: 164-168 (1985); Turner, A. D. et. al., "p-Amidino esters as irreversible inhibitors of factors IXa and Xa and thrombin", Biochemistry, 25: 4929-4935 (1986); and Sturzebecher, J. et. al., "Synthetic inhibitors of bovine factor Xa and thrombin. Comparison of their anticoagulant efficiency", Thromb. Res., 54: 245-252 (1989).
Unlike the reported polypeptide inhibitors of factor xa, the known small molecule inhibitors have been reported to be relatively non-selective with respect to the inhibition of other serine proteases. For example, 6-amidino-2-naphthyl-p-guanidinobenzoate dimethanesulfonate (FUT-175) inhibits human factor Xa and human thrombin similarly, yielding inhibitor constants of 4.1 .mu.M and 1.3 .mu.M, respectively. Hitomi et al., supra, at p. 166. p-Amidinophenyl .alpha.-methylcinnamate irreversibly inactivates human factor xa, human factor XIa and human thrombin similarly yielding second-order rate constants of inactivation of 9.9.times.10.sup.4, 16.times.10.sup.4, and 16.times.10.sup.4 M.sup.-1 min.sup.-1, respectively. Turner et al., supra, at p. 4932.